Method for oxidizing hydrocarbons with a hydroxylase from a methane monooxygenase

ABSTRACT

A purified hydroxylase component of the soluble methane monooxygenase enzyme present in the bacterium Methylosinus trichosporium OB3b is found capable of oxidizing hydrocarbons under aerobic conditions in the presence of suitable reducing agents. The hydroxylase can be reduced by commercial reducing agents, such as sodium dithionite and photo- and electrochemical means when in the presence of electron transport components, such as methyl viologen and proflavin. The hydroxylase can also be activated by hydrogen peroxide in the absence of reducing agents and molecular oxygen and is capable of oxidizing hydrocarbons under aerobic and anaerobic conditions in this manner. The hydroxylase component can be obtained with high final specific activity when ferrous iron compounds and cysteine are included in the purification buffers used to extract the hydroxylase from bacterial cells.

This is a continuation-in-part application to application Ser. No.600,575 filed Oct. 8, 1990 which is a continuation-in-part toapplication Ser. No. 352,721, filed May 16, 1989 now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to novel purified hydroxylase componentsof methane monooxygenase enzymes found in methanotrophic bacteria, anovel process for purifying the hydroxylases and a novel process foroxidizing hydrocarbons using purified hydroxylase, molecular oxygen (O₂)and commercially available reducing agents.

Alkanes are among the most unreactive carbon compounds. Alkane reactionshave characteristically high activation energies and often form productswhich are more reactive than the parent hydrocarbons. The reactions aredifficult to control for selective production of intermediate and endproducts in high yield. The oxidation of methane is an example of thedifficulties inherent in alkane reactions.

Methane reactions are of particular interest because large quantities ofnatural gas are located in remote areas, far from their main centers ofconsumption. It is expensive, however, to transport the gas to mostlocations where it is used. Current technology for methane conversion isbased on steam reforming, followed by either the Fischer-Tropsch processor by methanol synthesis. It is hoped that more direct paths to eitherproduct--methanol or higher hydrocarbons--can be found which are moreefficient, more selective and less costly. Other alkanes are alsoabundant and relatively inexpensive. New technologies would enable theseand other hydrocarbons to be used as inexpensive feedstocks for thesynthesis of commodity chemicals as well as precursors to liquid fuels.

Additionally, the increasing presence of hazardous substances such asbenzene, p-xylene and substituted hydrocarbons such as trichloroethylenein waste water streams demonstrates a need for a means for convertingthese substances into other substances which are less hazardous or nothazardous at all. As a result, researchers have looked to a variety ofmeans for converting hydrocarbons into other useful products.

In particular, many researchers have attempted to mimicnaturally-occurring biological systems. Certain bacteria have been foundwhich rely exclusively on methane as their source of life-sustainingcarbon compounds and energy. The first and most difficult step in theprocessing of methane by these methanotrophic bacteria is its conversioninto methyl alcohol. This conversion of methane to methanol is catalyzedby a family of enzymes now known as methane monooxygenases.

Methane monooxygenases utilize molecular oxygen as their oxygen source.Moreover, although methane is the only hydrocarbon known to sustaingrowth of the bacteria, methane monooxygenases are able to catalyze theoxidation of numerous saturated and unsaturated hydrocarbons. Oxidationis accomplished by forming an activated oxygen:enzyme:substrate complexcharged with two electrons from a suitable donar, such as NADH. The ironin methane monooxygenases is present in the form of a μ-oxo bridgedbinuclear iron center. Thus, the monooxygenases have two irons availablefor reaction. Other than the methane monooxygenases, no other proteinscontaining oxo bridged iron are known to catalyze oxygenase reactions.

Methanotrophic bacteria which utilize methane monooxygenases areclassified as Type I or Type II based on morphological differences intheir membrane-fine structure and divergence in their metabolicpathways. Both Type I and II methanotrophs are able to express methanemonooxygenases that are either soluble or membrane bound. Themonooxygenase that is expressed is determined by the conditions employedfor bacterial growth. Little is known about the membrane-boundmonooxygenases, but some of the soluble enzymes have been isolated andexamined. Examples include the methane monooxygenases isolated from theType I organism, Methylococcus capsulatus (Bath); the Type II organisms,Methylobacterium organophilum and Methylobacterium sp. (CRL-26); and theType II organism, Methylosinus trichosporium OB3b, the study of whichled to the present invention. Those examined to date appear to besimilar in composition and function.

Most methane monooxygenases isolated to date are comprised of threeproteins which are nominally designated in the literature as componentsA, B and C. Components A and C are also referred to as hydroxylase andreductase components, respectively, because of the roles they areperceived to play in bacterial oxidation. While others have isolated thecomponents from various strains of methanotrophic bacteria, thoseisolated prior to the present invention are characterized by lowspecific activities. Woodland et al., for example, have reported a finalspecific activity of 72 nmol/min/mg for the hydroxylase from the Type Iorganism, Methylococcus capsulatus (Bath) (J. Biol. Chem. 259, 53-59,1984); and Patel et al. have reported a final specific activity of 208for the hydroxylase from the Type II organism, Methylobacterium sp.(CRL-26) (J. Bact. 169, 2313-2317, 1987). These specific activities aremuch less than the approximately 800 nmol/min/mg specific activityobserved for in vivo oxidation of methane.

Methane monooxygenases appear to catalyze hydrocarbon oxidation in anorderly manner. Normally, the hydrocarbon to be oxidized, oxygen anddonated electrons collect at various sites of the monooxygenase systembefore oxidation occurs. More particularly, the reductase is believed toaccept donated electrons. Component B is believed to mediate thetransfer of electrons from the reductase to the hydroxylase where theoxidation is believed to occur. Previous studies by Dalton and Woodlandhave suggested that the substrate binds to the hydroxylase (Adv. Appl.Micro. 26, 71-87, 1980; J. Biol. Chem. 259, 53-59, 1984). The suggestionremained unconfirmed, however.

All three components are reportedly required for bacterial oxidation forMethylococcus capsulatus (Bath) (Colby et al., Biochem, J. 177, 903-908,1979) and Methylosinus trichosporium OB3b (Fox et al., Biochem. Biophys.Res. Comm. 154, 165-170, 1988). Component B is reportedly not requiredfor bacterial oxidation by Methylobacterium sp. (CRL-26) (Patel et al.,J. Bact. 169, 2313-2317, 1987; Patel, Arch. Biochem. Biophys. 252,229-236, 1987) and Methylobacterium organophilum (U.S. Pat. No.4,587,216 to Patel et al.). Patel has disclosed the use of purifiedhydroxylase and reductase proteins from various organisms in combinationwith a cofactor system comprising NADH or NADPH for oxidizinghydrocarbons (U.S. Pat. No. 4,587,216).

Some researchers have attempted to mimic oxidation by methanemonooxygenases using model compounds containing binuclear iron groups.Vincent et al., for example, have disclosed the oxidation ofhydrocarbons using the model complex Fe₂ O(OAc)₂ Cl₂ (bipy)₂ where bipyis 2,2'-bipyridine (J. Am. Chem. Soc. 110, 6898-6900, 1988). Oxidationwas accomplished using Bu^(t) OOH and, alternatively, O₂ as themonooxygen transfer reagent. The latter system also employed Zn powderand glacial acetic acid as electron and proton donors. Kitajima et al.have used synthetic analogues of hemerythrin to oxidize hydrocarbons (J.Chem. Soc. Chem. Comm. 7, 485-486, 1988). O₂ was used as the oxygensource in the presence of Zn powder and glacial acetic acid. Murch etal. have used (Me₁ N) [Fe₂ L(OAc)₂ ]⁵ where L isN,N'-(2-hydroxy-5-methyl-1,3-xylene)bis(N-carboxymethylglycine) (J.Amer. Chem. Soc., 108, 5027-5028, 1986). H₂ O₂ was used as the oxygensource. However, the oxidation rates obtained using these systems arelow and none have been shown capable of large scale oxidation ofhydrocarbons.

Although native methane monooxygenase systems are able to oxidizehydrocarbons, they do not present viable commercial alternatives to themore conventional methods for oxidation. Native systems obtain theelectrons needed for oxygen activation from such biochemicals as NADHand NADPH which are labile and expensive and, therefore, unsuitable forproduction at a commercial scale. Since most native systems comprisethree protein components which must be bound together, they are also toocomplex for efficient operation at commercial scale. Similarly, thetwo-component system disclosed by Patel also uses expensive and labilebiochemicals. Moreover, even the use of two components presentsdifficulties for oxidations at commercial scale.

Accordingly, it is an object of the present invention to provide a novelmethod for extracting novel purified hydroxylase components with highspecific activity from soluble methane monooxygenases.

It is another object of the present invention to provide a novel methodfor oxidizing hydrocarbons. More particularly, it is an object of thisinvention to provide a novel method for oxidizing hydrocarbons usingmolecular oxygen, chemical reductants or electrochemical orphotochemical means for supplying electrons in the presence of anelectron transfer-mediating compound, such as methyl viologen, and thepurified hydroxylases obtained from the soluble methane monooxygenase.

It is still another object of the invention to provide a novel methodfor oxidizing hydrocarbons in the presence of purified hydroxylase fromthe soluble methane monooxygenase using hydrogen peroxide as anoxidizing agent providing reduced oxygen to the reaction system.

It is still another object of this invention to provide a novel methodfor oxidizing hydrocarbons using purified, high activity hydroxylasefrom the methanotroph Methylosinus trichosporium OB3b, proflavin, methylviologen and sodium dithionite in the absence of reductase and componentB proteins.

Other objects, advantages and novel features of the invention will beapparent from the Description and Figures below.

SUMMARY OF THE INVENTION

As a class, proteins containing μ-oxo bridged iron groups have not beenknown to catalyze oxidation reactions. The oxidation of hydrocarbonscatalyzed by methane monooxygenases represents a new role for μ-oxobridged iron groups. The invention disclosed herein, therefore, relatesto a new method for catalyzing the oxidation of hydrocarbons. Moreparticularly, the invention comprises the use of hydroxylase proteinsfrom methanotrophic bacteria possessing μ-oxo bridged iron groups andcommercially available reducing agents to oxidize hydrocarbons. Theinvention further comprises the use of novel purification protocols forobtaining novel high activity hydroxylases.

Known biochemical processes for oxidizing hydrocarbons are believed toproceed in an orderly, step-wise manner. As discussed in Background,oxidation by methane monooxygenases proceeds by the step-wise formationof a oxygen:enzyme:substrate complex charged with two electrons from asuitable donor before the oxidation takes place. In the three-componentmethane monooxygenase systems, the electrons are believed to be firstaccepted by the reductase component and then transferred to thehydroxylase component where the oxidation actually takes place. Theelectron transfer is believed to be mediated by the B component.

It has been found, however, that purified hydroxylase component can beseparated from the monooxygenase system and used to catalyze theoxidation of hydrocarbons in the absence of the reductase and Bcomponents of the monooxygenase system. It has also been found that thekey functional group of the hdyroxylase for catalysis is a μ-oxo bridgediron group, that both irons in the bridged group must be in the reduced(ferrous) form for catalysis to occur, and that the hydroxylase can bebrought to the fully reduced form by chemical reducing agents (e.g.,sodium dithionite) and electrochemical (e.g., electrochemical cell) andphotochemical means (e.g., bright light from a projection lamp) forsupplying electrons, when in the presence of a suitable electrontransfer-mediating compound, such as methyl viologen. The novel processfor oxidizing hydrocarbons of the present invention comprises contactingthe purified hydroxylase of a soluble methane monooxygenase enzyme withthe hydrocarbon to be oxidized in the presence of an electron source inthe presence of electron transfer-mediating compound, such as methylviologen. The novel process for oxidizing hydrocarbons is able tooxidize alkanes, alkenes, aromatic hydrocarbons, substitutedhydrocarbons and mixtures thereof. The hydrocarbons methane, propane,benzene, propene and trichloroethylene are preferred substrates.

In addition, it has been found that purified hydroxylase separated fromthe monooxygenase system can be used to catalyze the oxidation ofhydrocarbons simply in the presence of hydrogen peroxide without the useof chemical reducing agents or electron sources, an electrontransfer-mediating compound, or molecular oxygen. This embodiment of thenovel process comprises contacting the purified hydroxylase of a solublemethane monooxygenase enzyme with the hydrocarbon to be oxidized in thepresence of hydrogen peroxide. The novel process is effective inoxidizing a range of hydrocarbons and affords the additional advantagesthat oxygen and reducing equivalents, both of which are contained in theperoxide, are provided to the hydroxylase in a single step, and theoxidation can occur under anaerobic as well as serobic conditions.

It has also been found that the hydroxylase component can be purified toparticularly high specific activity and that this high activity can beattributed to high iron concentration within the hydroxylase. The highactivity hydroxylase of the present invention has a final specificactivity of at least about 800 nmol/min/mg. The hydroxylase typicallyhas an iron concentration of at least about 3.5 mols iron per molhydroxylase. A hydroxylase with a final specific activity of at leastabout 1000 nmol/min/mg is preferred. The preferred hydroxylase typicallyhas an iron concentration of at least about 4.0 mols iron per molhydroxylase.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

A soluble three-component methane monooxygenase from the Type IImethanotroph Methylosinus trichosporium OB3b was purified tohomogeneity. The novel purification protocols permitted preservation ofmost of the activity present in the intact organism throughout thepurification procedure. All methane monooxygenase components wererecovered in high yield and exhibit much higher activities than havebeen reported previously. The purified hydroxylase is capable ofsupporting oxidation of alkanes (including methane), alkenes, aromaticand substituted hydrocarbons in the absence of the other twomonooxygenase components.

Relationship of the Stabilizers to Activity--The novel high activityhydroxylase of the present invention was obtained from Methylosinustrichosporium OB3b using a novel purification protocol. The protocolmakes use of stabilizers which have been used in the purification ofother oxygenases but which had never been used in the purification ofmethane monooxygenases. The stabilizers comprise ferrous iron (Fe²⁺)containing compounds and cysteine. The stabilizers provide a source ofiron for the purified hydroxylase that is otherwise lost duringpurification and facilitates retention of the structural integrity ofthe hydroxylase as well. Ferrous ammonium sulfate is the preferred ironcontaining compound. Addition of these stabilizers to buffer solutionsused in the purification of the hydroxylase results in significantimprovement in the specific activity of the hydroxylase as well as anincrease in iron content from ˜2 irons per hydroxylase as previouslyobserved to ˜4 as is routinely observed in the most active hydroxylasepreparations of the present invention.

The Role of the Hydroxylase Metal Center--The hydroxylase contains aspin-coupled binuclear iron cluster with a bridging oxo or hydroxoligand (Fox et al., J. Biol. Chem. 263, 10553-10556, 1988; Woodland etal., Biochim. Biophys. Acta 873, 237-242, 1986; Prince et al., Biochim.Biophys. Acta 952, 220-229, 1988; Ericson et al., J. Amer. Chem. Soc.110, 2330-2332, 1988). Single turnover and EPR results show that themixed valent state (Fe²⁺ -Fe³⁺) is unreactive toward O₂ on the timescale of the catalytic process. This also appears to be the case for themixed valent state of the hydroxylase from Methylococcus capsulatus(Bath) and other proteins.

Relationship of the Hydroxylase Iron Center to Activity--Previouspreparations of the hydroxylase from other bacteria were reported tocontain approximately one binuclear iron cluster per mol (Woodland etal., J. Biol. Chem. 259, 53-59, 1984; Patel et al., J. Bact. 169,2313-2317, 1987). Similarly, low activity preparations of theMethylosinus trichosporium OB3b hydroxylase presented here contained0.8-1 cluster per mol on average. However, as indicated in Table III,high activity preparations of the OB3b hydroxylase containedsubstantially greater amounts of iron clusters per mol hydroxylase. Inthe most extreme case, an increase of approximately 2-fold in the ironcluster concentration corresponded to an increase in specific activityof about 25-fold. Since iron is present in the high activity hydroxylaseonly in the form of a binuclear iron cluster, this strongly implies thatthe iron clusters are involved in catalysis (Fox et al., J. Biol. Chem.263, 10553-10556, 1988).

A novel high activity hydroxylase of the present invention was purifiedand characterized as follows:

Bacterial Growth--Methylosinus trichosporium OB3b was provided by Dr. R.S. Hanson (University of Minnesota) and maintained under methane and air(1:3 v/v) as described by Cornish et al. (J. Gen. Micro. 130, 2565-2575,1984). The concentration of FeSO₄.7H₂ O was increased to 80 μM duringlarge scale growth. The cells were grown at 28°-32° C. in continuousculture (dilution=0.05 h⁻¹). Commercial grade methane from Air Products,Allentown, Pa., was used as the sole carbon source. A New BrunswickCFS-314 fermentor vessel was used and sparged at the following rates:methane, 600-1500 ml/min, 15 psig; air, 2500-4500 ml/min, 15 psig. Cellswere harvested using an HPK 40 Pellicon cassette system equipped with 5ft² of 0.45μ HVLP membrane (Millipore Corp., Bedford, Mass.), washedwith cold 20 mM sodium phosphate buffer, pH 7, and centrifuged at 9000×gfor 20 min. The cell paste was stored at -80° C. Typically, 18-25 g ofcell paste per liter of culture media gave high enzyme activity.

Enzyme Assays--Assays of bacterial cells were performed at 30° C. usingcells harvested directly from the fermentor vessel. Methane oxidationwas measured by gas chromatography by monitoring depletion of methanefrom the headspace of 30 ml reaction vials. Oxygen consumption wasmeasured polarographically as described by Arciero et al. (J. Biol.Chem. 258, 14981-14991, 1983). The bacterial suspension was supplementedwith 100 μM formaldehyde before gas chromatographic assays forconversion of propene to propene oxide in order to allow adequateregeneration of NADH by endogenous formaldehyde and formatedehydrogenases. Gas chromatographic and polarographic assays of themethane monooxygenase system were performed as described by Fox et al.(Biochem. Biophys. Res. Comm. 154, 165-170, 1988). Unless explicitlystated, the specific activity values reported for the enzyme componentsare those observed during the hydroxylation reaction in the presence ofoptimal concentrations of the other components of the methanemonooxygenase. The optimal concentrations were determined by varying theconcentration of one component relative to fixed concentrations of theother two components. In routine polarographic assays of methanemonooxygenase activity, furan is an acceptable substrate withcharacteristics which facilitate the assay. Furan is a water solubleliquid (64 mM at 30° C.) which is highly susceptible to oxidation. Forthe complete methane monooxygenase, furan has K_(M)(app) =20 μM andturnover number=7.6 s⁻¹ relative to the hydroxylase. (For purposes ofthe present invention, turnover number is the number of catalytic cyclesper second per molecule.) Solutions of furan were standardized by gaschromatography as described by Nicholson et al: (Anal. Chem. 49,814-819, 1977). Column fractions were screened for reductase activity byobserving the catalyzed reduction of 2,6-dichlorophenol indophenol inthe presence of NADH at 600 nm (ε₆₀₀ =13 mM⁻¹ cm⁻¹) (Hultquist, D. E.,Meth. Enz. 52, 463-473, 1978). A typical assay contains 50 mM MOPS(3-(N-morpholino)propane sulfonic acid), pH 7, 100 μM 2,6-dichlorophenolindophenol, 1 mM NADH, and 0.3-3 nmol reductase in a total volume of 1ml.

Preparation of the Cell Free Extract--The cell paste (200 g) wassuspended in 200 ml of 25 mM MOPS, pH 7.0, containing 200 μM Fe(NH₄)₂(SO₄)₂.6H₂ O, and 2 mM cysteine (buffer A). The cells were sonicated atintervals for a total of 16 min with sufficient cooling to maintain thetemperature below 4° C. The sonicated suspension was diluted with anadditional 200 ml of buffer A and centrifuged at 48,000×g for 90 min.The supernatant was carefully decanted, diluted with an additional 200ml of buffer A, and adjusted to pH 7.0. The diluted supernatant iscalled the cell free extract.

Separation of the Components of the Methane Monooxygenase--The cell freeextract was immediately loaded onto a fast flow DEAE Sepharose CL-6Bcolumn (40 mm×250 mm) equilibrated with freshly prepared buffer A at alinear flow rate of 40 cm/h. After loading, the column was washed withan additional 600 ml of buffer A. All methane monooxygenase componentswere completely adsorbed under these conditions. The methanemonooxygenase components were eluted with a 2 l gradient of 0.0-0.40MNaCl in buffer A at a linear flow rate of 15 cm/h. Fractions containingthe hydroxylase, component B, and the reductase eluted at 0.075M NaCl,0.18M NaCl, and 0.27M NaCl, respectively. Table I is a summary of thepurification of the methane monooxygenase into its components.

Purification of the Hydroxylase--Pooled hydroxylase fractions wereimmediately concentrated by ultrafiltration. The concentrated proteinwas applied immediately to a Sephacryl S-300 column (40 mm×950 mm)equilibrated in 25 mM MOPS, pH 7.0 containing 100 μM Fe(NH₄)₂ (SO₄)₂.6H₂O, 2 mM cysteine, 1 mM dithiothreitol, and 5% (v/v) glycerol at a linearflow rate of 6 cm/h. Hydroxylase fractions were pooled, concentrated viaultrafiltration, frozen in liquid nitrogen, and stored at -80° C.

Purification of Component B--Solid (NH₄)₂ SO₄ was added to the pooledcomponent B fractions to make a 50% saturated solution at 4° C. After 20min, the solution was centrifuged at 30000×g for 30 min. The pellet wassuspended in 25 mM MOPS, pH 7.0 and applied to a Sephadex G-50 column(40 mm×950 mm) equilibrated in the same buffer at a linear flow rate of3 cm/h. The pooled fractions of component B were then applied to a fastflow DEAE Sepharose CL-6B column (23 mm×80 mm) equilibrated in 25 mMMOPS, pH 6.5. Component B was eluted with a 500 ml gradient from0.08-0.25M NaCl in the same buffer at a linear flow rate of 4 cm/h.Component B fractions were pooled, concentrated via ultrafiltration,frozen in liquid nitrogen, and stored at -80° C.

Purification of the Reductase--Pooled reductase fractions were dilutedwith an equal volume of 25 mM MOPS, pH 6.5 containing 5 mM sodiummercaptoacetic acid. The solution was then adjusted to pH 6.5 andapplied to a fast flow DEAE Sepharose CL-6B column (23 mm×80 mm)equilibrated in the same buffer. The column was washed with 100 ml ofequilibration buffer containing 0.15M NaCl at a flow rate of 30 cm/h. A500 ml gradient from 0.15-0.32M NaCl was then applied to the column at alinear flow rate of 5 cm/h. The pooled fractions were concentrated usinga fast flow DEAE Sepharose CL-6B column (6 mm×15 mm). The concentratedreductase was applied to an Ultrogel AcA-54 column (25 mm×450 mm)equilibrated in 25 mM MOPS, pH 7.0, containing 5 mM mercaptoacetic acidand eluted at a linear flow rate of 3 cm/h. Fractions exhibiting aconstant A₄₅₈ /A₃₄₀ ratio of 1.3 and hydroxylation activity greater than20% of the peak fraction were pooled, concentrated using DEAE SepharoseCL-6B, frozen in liquid nitrogen, and stored at -80° C.

Molecular Weight Determinations--Ultracentrifugation experiments wereperformed using a Beckman Model E Series 400 ultracentrifuge with anAn-D rotor using schlieren optical visualization. Sedimentationequilibrium experiments were performed according to the method ofmeniscus depletion as described by Yphantis (Biochemistry 3, 297-317,1970). The following conditions were used for meniscus depletionexperiments: hydroxylase (protein concentration 0.6 mg/ml, density 1.01g/ml, viscosity 1.0 ml/g, estimated v_(o) 0.73, 15,220 rpm, 12.5° C.);component B (protein concentration 1.5 mg/ml, density 0.9997 g/ml,viscosity 1.0 ml/g, estimated v_(o) 0.73, 20,410 rpm, 12.4° C.);reductase (protein concentration 1.7 mg/ml, density 1.004 g/ml,viscosity 1.0 ml/g, estimated v_(o) 0.74, 12590 rpm, 10.5° C.).Sedimentation velocity experiments were performed as described inSchachman (Meth. Enz. 4, 32-103, 1957). The following conditions wereused for sedimentation velocity experiments: hydroxylase (proteinconcentration 3.33 mg/ml, density 1.01 g/ml, viscosity 1.0 ml/g,estimated v_(o) 0.73, 25,600 rpm, 6° C.); component B (proteinconcentration 2.2 mg/ml, density 0.9997 g/ml, viscosity 1.0 ml/g,estimated v_(o) 0.73, 44,800 rpm, 6° C.); reductase (proteinconcentration 1.6 mg/ml, density 1.004 g/ml, viscosity 1.0 ml/g,estimated v_(o) 0.74, 25,000 rpm, 6° C.). The partial specific volumevalues were determined from amino acid analysis by the Department ofBiochemistry, University of Minnesota. Native molecular weights andestimation of the Stokes radii were also determined by gel filtration(Yamaguchi et al., J. Biol. Chem. 253, 8848-8853, 1978). Denaturingpolyacrylamide gel electrophoresis was done as reported in Laemmli(Nature 227, 680-685, 1970).

Other Methods--Protein concentrations were determined colorimetricallyusing dialyzed and lyophilized samples of the purified proteins asstandards as reported by Bradford (Anal. Biochem. 72, 248-254, 1976).The concentrations of the purified proteins were also determined usingquantitative amino acid analysis from which extinction coefficients at280 nm were calculated. Iron was determined spectrophotometrically bycomplexation with 2,4,6-tripyridyl-s-triazine (Fischer et al., Clin.Chem. 10, 21-25, 1964). Iron and other metals were also determined byinductively coupled plasma emission spectroscopy by the Soil ScienceServices, University of Minnesota, St. Paul. Flavin identity and contentwere determined by reverse phase high pressure liquid chromatographywith a 0 to 50% methanol gradient in 5 mM ammonium acetate, pH 6.0(Hausinger et al., Meth. Enz. 122, 199-209, 1986). Flavin content wasalso determined optically (ε₄₅₀ =11.3 mM⁻¹ cm⁻¹) after precipitation ofthe reductase using trichloroacetic acid. Inorganic sulfide wasdetermined by the method of Beinert (Anal. Biochem. 131, 373-378, 1983).Hydrogen peroxide and O₂ ⁻ were determined by catalase or superoxidedismutase coupled O₂ evolution, respectively. Chromatofocusing wasperformed using Polybuffer exchanger PBE94 with Polybuffer 74(Pharmacia, Piscataway, N.J.). Protein molecular weight standards, MES,MOPS, NADH, dithiothreitol, cysteine, phenazine methosulfate, proflavin,and methyl viologen were obtained from Sigma (St. Louis, Mo.). Residualethanol (an inhibitor of the hydroxylation reaction) was removed fromNADH by repeated evaporation from MOPS buffer. All other chemicals wereof the best quality available and were used without furtherpurification.

Purified Methane Monooxygenase Components--Methane monooxygenaseactivity is reproducibly observed in cell free extracts of Methylosinustrichosporium OB3b prepared as described above. This activity is fullysoluble and represents all of the methane monooxygenase activityobserved in the intact organisms. The activity of the cell free extractvaries significantly from preparation to preparation suggesting that thecells are very sensitive to growth conditions. The overall specificactivity was found to range from 20-80 nmol/min/mg when assayed withoutthe addition of supplemental methane monooxygenase components. By addingoptimal amounts of two of the purified methane monooxygenase componentswhile assaying for the third component in the cell free extract, thespecific activities of the single components are found to be 20-80nmol/min/mg for the reductase and 50-200 nmol/min/mg for the hydroxylaseand component B. Each of the components have been purified in high yieldand high specific activity as described above. The results ofrepresentative purifications of the three methane monooxygenasecomponents are summarized in Table I. The physical data obtained for thecomponents are shown in Table II, where analogous data for thepreviously reported soluble methane monooxygenase systems is alsopresented for comparison.

Properties of the Hydroxylase--Physical properties of all threemonooxygenase components are presented in Table II. The hydroxylaseobtained by the two-step chromatographic procedure shown in Table I ispure as judged by repeated gel filtration, ultracentrifugation, anddenaturing gel electrophoresis. In particular, there is no evidence forcontamination by either of the two other monooxygenase components. Forexample, antibodies were raised to the purified component B andreductase, but were not observed to cross-react with the purifiedhydroxylase preparations. The molecular weight determined by analyticalultracentrifugation is 245 kDa. Native gel electrophoresis indicatesthat a single protein component is present in the purified samples.Denaturing gel electrophoresis shows that the protein consists of threesubunit types with molecular weights of about 54.4, 43.0, and 22.7 kDa,suggesting that the quaternary structure is (αβγ)₂ as observed for allother purified methane monooxygenase hydroxylase components. As shown inTable II, the maximal specific activity of the hydroxylase isolated fromMethlyosinus trichosporium OB3b using the procedures described above(1700 nmol/min/mg) is 8-25 times higher than the specific activities ofhydroxylase components purified from other methanotrophs.

The oxidized form of the hydroxylase exhibits an electronic absorptionmaximum at 282 nm and a very weak absorption which decreases smoothlythrough the visible region. No distinct features are observed above 300nm. Similarly, no optical spectrum above 300 nm is observed for eitherthe partially or the fully reduced forms of the hydroxylase. Otherhydroxylase preparations have been reported by Woodland et al. (J. Biol.Chem. 259, 53-59, 1984) and Patel et al. (J. Bact. 169, 2313-2317,1987). In contrast to these other preparations, no absorption isobserved in the regions near 410 nm and 550 nm in any oxidation state.In other proteins containing μ-oxo bridged iron clusters, absorbancemaxima near 410 and 550 nm have been associated with tyrosine radicalformation and tyrosine ligation, respectively (Atkin et al., J. Biol.Chem. 248, 7464-7472, 1973; Averill et al., J. Amer. Chem. Soc. 109,3760-3767, 1987). The 8.5-fold increase in specific activity obtainedduring the purification indicates that the hydroxylase from Methylosinustrichosporium OB3b comprises roughly 12% of the soluble protein, anumber supported by the 8% recovery of total protein as hydroxylase.Inductively coupled plasma emission spectroscopy performed on thepurified hydroxylase indicates that iron is the only metal present instoichiometric amounts or greater.

Properties of the Reductase--The monomeric reductase from Methylosinustrichosporium OB3b has a molecular weight of about 39.7 kDa as measuredby analytical ultracentrifugation, quantitative gel chromatography anddenaturing gel electrophoresis. The reductase was found to contain 1 molFAD per mol reductase. The ratio of iron to inorganic sulfide (Table II)and the optical spectral properties are consistent with the presence ofa [2Fe-2S] cluster as has been observed for the reductase purified fromother bacteria (Colby et al., Biochem. J. 177, 903-908, 1979; Patel, R.N., Arch. Biochem. Biophys. 252, 229-236, 1987). The reductase has aspecific activity of 26100 nmol/min/mg for the hydroxylation reactioncatalyzed by the complete methane monooxygenase. This value isapproximately four times that reported for the reductase purified fromother methanotrophs. The purified reductase also catalyzes the reductionof 2,6-dichlorophenol indophenol at pH 7.0 with a specific activity of127 μmol/min/mg. The yield and the fold purification obtained suggestthat the reductase accounts for approximately 0.1 to 0.3% of the solubleprotein present in the cell free extract. Thus, on a molar basis, thereductase is present in the cell at about 10% of the concentration ofthe other two components.

Properties of Component B--Component B from Methylosinus trichosporiumOB3b is a monomeric protein of about 15.8 kDa molecular weight asdetermined by analytical ultracentrifugation and denaturing gelelectrophoresis. Component B has no visible spectrum above 300 nm. Theyield and the fold purification obtained suggests that component Baccounts for approximately 0.5% of the soluble protein. On a molarbasis, component B and the hydroxylase appear to be present inapproximately equal amounts. The purification of component B shown hereresults in a 5-fold increase in yield and 1.5-fold increase in specificactivity over the comparable protein from Methylococcus capsulatus(Bath) (Green et al., J. Biol. Chem. 260, 15,795-15,801, 1985). Theother physical properties shown in Table II are quite similar to thecorresponding protein from Methylococcus capsulatus (Bath) (Green etal., J. Biol. Chem. 260, 1579-15,801, 1985).

Correlation of the Oxo Bridged Iron Cluster with Activity--The novelhigh activity hydroxylase of the present invention is furthercharacterized by high iron concentration at the active site of thehydroxylase. The measurement of total iron content of the hydroxylasedoes not distinguish between iron present at the active site in abinuclear cluster and iron adventitiously bound to the hydroxylasesurface. However, this distinction can be readily made using Mossbauerand EPR spectroscopy. Mossbauer spectroscopy provides a direct estimateof the iron present in the oxidized state of the binuclear iron clusterthrough quantitation of the characteristic quadrupole doublet observedat 4.2° K. (Fox et al., J. Biol. Chem. 263, 10,553-10,556, 1988).Mossbauer spectroscopy also allows the relative amount of iron presentin each of the three possible redox states of the cluster to bedetermined with reasonable accuracy. Likewise, quantitation of the EPRsignals from the mixed valent and fully reduced states of the binuclearcluster allows reasonable estimates of the concentration of clusterpresent in these states (Fox et al., Biophys. Biochem. Res. Comm. 154,165-170, 1988; Fox et al., J. Biol. Chem. 263, 10,553-10,556, 1988).Since the mixed valent state must be produced by titration, the valuesreported in Table III for the mixed valent state are somewhat variable.Hydroxylase preparations exhibiting a 25-fold range of specific activityvalues have been studied. Both Mossbauer and EPR spectroscopicmeasurements show that the binuclear cluster concentration increases inthe most active preparations of the hydroxylase. Moreover, thesepreparations contain greater than one binuclear cluster per mol.However, it is also clear that a wide range of specific activities arepossible with little change in cluster concentration especially forsamples with specific activities less than 500.

Catalysis Via the Hydroxylase

The novel process for oxidizing hydrocarbons of the present inventionobtains from the discovery that the hydroxylase of the methanemonooxygenase from Methylosinus trichosporium OB3b is able to catalyzethe oxidation of hydrocarbons in the absence of the reductase and Bcomponents of the native enzyme. As demonstrated by EPR spectroscopy,the binuclear iron cluster of the hydroxylase is in the fully reduced(diferrous) state when it is catalytically competent. The hydroxylasecan be brought to the fully reduced state by chemical reductants (e.g.,sodium dithionite) and photochemical and electrochemical means whichprovide electrons needed to reduce the hydroxylase, and compounds whichare able to transfer the electrons to the hydroxylase to convert it tothe fully reduced state. Methyl viologen and proflavin, for example, areable to mediate the transfer of electrons to the hydroxylase.

The hydroxylase can also be brought to a catalytically competent stateby hydrogen peroxide. The use of hydrogen peroxide affords additionaladvantages in that the reducing equivalents and the oxygen required foroxidation are provided directly to the hydroxylase. Accordingly, noelectron transport mediating compounds are required and no additionaloxygen source is required. This provides the further advantage that,oxidation can occur under anaerobic as well as aerobic conditions.

The isolated hydroxylase was able to oxidize various hydrocarbons,including alkanes, alkenes, aromatic and substituted hydrocarbons in thepresence of sodium dithionite, methyl viologen and proflavin.Experimental conditions and results were as follows:

Single Turnover Oxidation of Propane and Propene--Samples of highactivity hydroxylase (1000 nmol/min/mg, 100 nmol protein) in 100 mMMOPS, pH 7.0 were supplemented with 100 μM phenazine methosulfate forconversion to the mixed valent state, or with 10 μM proflavin and 100 μMmethyl viologen for conversion to the fully reduced state. The mixedvalent samples were prepared in EPR tubes under anaerobic conditions andthe fully reduced samples were prepared in Teflon sealed 3 ml reactionvials. Anaerobiosis was established by repeated cycles of evacuation andflushing with Ar gas that had been made oxygen-free by passage throughan activated copper oxygen scrubbing trap (BASF, Inc.) as described inArciero et al. (J. Biol. Chem. 258, 14,981-14,991, 1983). For the mixedvalent samples, reduction was performed by addition of one equivalent ofsodium dithionite (two reducing equivalents, based on the presence oftwo binuclear iron clusters) to the hydroxylase, while for the fullyreduced samples, two equivalents of sodium dithionite (four reducingequivalents, based on the presence of two binuclear iron clusters) wereadded. After addition of sodium dithionite, the samples were incubatedfor 5 min at 30° C. No residual blue color indicative of the presence ofmethyl viologen radical (ε₆₀₄ =14.4M⁻¹ cm⁻¹) was observed, showing thatexcess sodium dithionite was not present. The extent of reduction in themixed valent samples was monitored by EPR spectroscopy. Aftermeasurement of the EPR spectrum, the mixed valent samples weretransferred anaerobically from the EPR tube to a 3 ml reaction vialfilled with Ar. Propane or propene (3 ml) was added to the reaction vialand allowed to equilibrate for 5 min at 30° C. The reaction was startedby injection of 3 ml of air into the reaction vial, followed by rapidmixing of the solution. After an appropriate time period (5 s orlonger), the reaction was terminated by injection of 100 μl ofchloroform followed by a brief period of vortexing. The chloroform layerwas separated by centrifugation (2 min at 10,800×g) and analyzed forreaction product by gas chromatography. 1-Propanol was obtained frompropane to a yield of 10.1% and propene was oxidized to propene oxide(40.1%) (see Table IV(A)).

Addition of component B or the reductase does not significantly changethe product yield, and these components are found to be incapable ofcatalyzing hydroxylation alone. Hydroxylase inactivated by heatprecipitation is incapable of catalyzing hydroxylation. This providesthe first evidence based on catalysis that the site of the monooxygenasereaction is located on the hydroxylase component. Single turnoverexperiments performed using the hydroxylase reduced only to the mixedvalent state as described above result in much lower yields of1-propanol or propene oxide from propane and propene, respectively(Table IV(A)). The yields obtained are approximately those expected fromthe concentration of fully reduced hydroxylase unavoidably present alongwith the mixed valent state; thus it is probable that only the fullyreduced hydroxylase is capable of hydroxylation. (This conclusion issupported by the observation that only the fully reduced hydroxylase israpidly oxidized by oxygen.)

The product of enzymic propane hydroxylation is exclusively 1-propanol,whether catalyzed by the hydroxylase alone under single turnoverconditions or by the reconstituted methane monooxygenase system. Noevidence for 2-propanol, the predominant product for hydroxylationcatalyzed by small molecule catalysts, was observed. (For example, themodel complex Fe₂ O(OAc)₂ Cl₂ (bipy)₂, where OAc is acetate and bipy is2,2'-bipyridine, catalyzes the hydroxylation of ethane, propane, andcyclohexane in the presence of t-butyl peroxide (Vincent et al., J. Am.Chem. Soc. 110, 6989-6900, 1988). The hydroxylation of propane isconducted under 90 psi propane for up to two days and yields 8.8%2-propanol.) Thus, the enzyme catalyzed reaction retains specificityunder single turnover conditions, strongly implying that the reaction iscatalyzed on the enzyme surface and that the reductase and component Bdo not play a role in directing the catalysis chemistry. The singleturnover hydroxylation reaction appears to be complete in less than 5 s,which was the minimum time in which the reactants could be mixed and thereaction stopped. Thus, the turnover number of the hydroxylase alonemust be at least 0.7 s⁻¹ and is probably much greater. This compareswell with the accurately determined turnover number of the completesystem of 4.4 s⁻¹ measured under biological turnover conditions.

Single Turnover Oxidation of Methane--Samples of low activityhydroxylase (˜160 nmol/min/mg, 340 nmol), methyl viologen (40 nmol) andproflavin (0.25 nmol) in 25 mM MOPS buffer, pH 7.5 were placed in a 3 mlreaction vial sealed with a Teflon septum and made anaerobic by repeatedcycles of evacuation and flushing with O₂ -free Ar. Methane gas was thenflushed through the sealed vial for 5 min. After a further 5 minincubation period to allow the equilibration of methane into the liquidphase, a three-fold excess of sodium dithionite relative to proteinconcentration was added to the hydroxylase solution. This solution wasallowed to incubate for 5 min at 23° C., and then 3 ml of air wasinjected into the vial. The contents of the vial were rapidly mixed.After˜10 min, the contents of the vial were placed in a Centriconconcentrator device with a YM30 membrane and spun for 30 min at 5000rpm. The aqueous phase that passed through this membrane was analyzedfor methanol using gas chromatography. The yield was 61 nmol methanol or18% based on protein concentration (see Table IV(B)).

Multiple Turnover Oxidation of Benzene--Samples of low activityhydroxylase (˜160 nmol/min/mg, 400 nmol), methyl viologen (320 nmol) andproflavin (0.5 nmol) were placed in a 3 ml reaction vial sealed with aTeflon septum and made anaerobic by repeated cycles of evacuation andflushing with O₂ -free Ar.

The following procedure was repeated for two cycles: After˜2 min,benzene (50 μmoles) was added to the reaction mixture, mixed gently andallowed to equilibrate with protein solution for 5 min. Then anequimolar amount of sodium dithionite relative to hydroxylaseconcentration was added to the hydroxylase solution. This solution wasallowed to incubate for 5 min at 23° C. and then 3 ml of air wasinjected into the vial. The contents of the vial were rapidly mixed. Thereaction mixture was extracted with CHCl₃ and analyzed for phenol usinggas chromatography. Phenol was formed at a yield of about 3.4% based onnmol phenol formed per nmol hydroxylase for each cycle (see TableIV(B)).

Multiple Turnover Oxidation of Propene--Samples of low activityhydroxylase (˜155 nmol/min/mg, 340 nmol), methyl viologen (40 nmol) andproflavin (0.25 nmol) and 50 μl of 0.5M MOPS buffer, pH 7.0 were placedin a 3 ml reaction vial sealed with a Teflon septum and made anaerobicby repeated cycles of evacuation and flushing with O₂ -free Ar. Thehydroxylase specific activity after this cycle was˜151.

The following procedure was repeated for seven cycles: After˜2 min, theheadspace of the vial was flushed for 2 min with propene gas at a flowrate of 2 ml/min. An equimolar amount of sodium dithionite relative tohydroxylase concentration was added to the hydroxylase solution. Thissolution was allowed to incubate for 5 min at 23° C., and then 3 ml ofair was injected into the vial. The contents of the vial were rapidlymixed. The reaction mixture was analyzed for propene oxide by gaschromatography. The total yield after seven cycles was 533 nmol propeneoxide or 156% based on protein concentration (see Table V). Thisrepresents about 1.6 turnovers per hydroxylase molecule present in thereaction mixture. These results also indicate that the hydroxylase cancatalyze repeated oxidations without inactivation of the enzyme. Asindicated in Table V the measured activity of the hydroxylase wasslightly greater after the second cycle, and the product yield increaseduntil the experiment was terminated after seven cycles.

Single Turnover Oxidation of Trichloroethylene--Low activity hydroxylase(210 nmol/min/mg, 100 nmol) and methyl viologen (40 nmol) intrichloroethylene-saturated 25 mM MOPS buffer, pH 7.5, were placed in a3 ml reaction vial sealed with a Teflon septum. A two-fold excess ofsodium dithionite relative to protein concentration was added to thehydroxylase solution. The contents of the vial were rapidly mixed,followed by the addition of 500 μl of benzene after 5 seconds. Thereaction products, including trichloroethylene oxide, were extractedinto benzene. Since trichloroethylene oxide is unstable, it was reactedwith 4-(p-nitrobenzyl)pyridine to form a stable derivative. The yieldwas determined by comparing the optical spectrum of the derivatizedproduct with the optical spectrum of the analogous derivative formedfrom synthetic trichloroethylene epoxide. The yield was about 6 to 8nmol trichloroethylene oxide or about 8% based on protein concentration.

The isolated hydroxylase is also able to oxidize hydrocarbons usingelectrochemical means as the source of reducing electrons while in thepresence an electron transporter, such as methyl viologen.

Electrochemical Oxidation of Propene--Low activity hydroxylase was usedto oxidize propene to propene oxide in the presence of electrochemicallyreduced methyl viologen with and without the additional electrontransfer mediator proflavin. Experimental conditions and results were asfollows:

A sample of low activity hydroxylase (155 nmol/min/mg, 342 nmol) in 25mM MOPS buffer, pH 7.5 was made anaerobic by repeated evacuation andreflushing with O₂ -free Ar gas. Methyl viologen was reduced at a goldelectrode under anaerobic conditions in an electrochemical cell andadded anaerobically to the hydroxylase system before each cycle untilthe blue color of reduced methyl viologen just persisted after a fewminutes of incubation. The system was flushed with propene after which 3ml of air was injected into the system. The contents were then rapidlymixed. The reaction mixture was analyzed for propene oxide by gaschromatography. The oxidation cycle was repeated twice more.

Propene oxide was detected as a reaction product after each cycle. Theyields for each cycle were 51, 122 and 140 nmol, respectively, and thetotal yield was 313 nmol of the epoxide. Based on protein content, thiswas 92%.

Propene was also oxidized to 1,2-propene oxide as above but with theaddition of proflavin to the system. The reaction system was prepared asabove using 548 nmol hydroxylase and 0.5 nmol proflavin. A single cycleoxidation was performed. The yield was 340 nmol epoxide or 62% based onprotein content.

The isolated hydroxylase is also able to oxidize hydrocarbons usingphotochemical means as the source of reducing electrons while in thepresence of an electron transporter, such as methyl viologen. Forexample, bright light will decarboxylate glycine. When methyl viologenis present during the decarboxylation, it will accept electrons to formmethyl viologen radicals. These radicals are able to transmit electronsto the hydroxylase thereby enabling it to oxidize hydrocarbons.

Photochemical Reduction of the Hydroxylase--Low activity hydroxylase(˜155 nmol/min/mg, 59 nmol) in 200 μl of 0.1M MOPS containing 0.1Mglycine and 1 mM methyl viologen was placed in a sealed EPR tube. Thetube was wrapped in aluminum foil to protect the contents from exposureto light. Oxygen was removed from the tube by repeated evacuation andflushing with O₂ -free argon gas as described above. EPR spectra of theoxygen-free sample showed no signal at g=15 indicating the hydroxylasewas in the oxidized state. The sample was then placed in a water bath at4° C. and exposed to bright light from a projection lamp. EPR spectra ofthe light irradiated sample showed the signal at g=15, characteristic ofthe fully reduced hydroxylase. The hydroxylase is able to oxidizehydrocarbons in this state.

The isolated hydroxylase was able to oxidize various hydrocarbons,including alkanes, alkenes and aromatic hydrocarbons in the presence ofhydrogen peroxide. Experimental conditions and results were as follows:

Multiple Turnover Oxidation of Hydrocarbon Substrates Using HydrogenPeroxide--Samples of high activity hydroxylase (˜880 nmol/min/mg, 25nmols) in 180 μl of 25 mM MOPS, pH 7.5 were placed in 3 ml conicalbottom reaction vials sealed with teflon-faced silicone septa and screwcap assemblies. Substrates were injected into the respective reactionvials as follows: gaseous substrates (3 ml) were injected into theheadspace of the reaction vial; liquid substrates (50 μls) were injecteddirectly into the protein solution. The protein solution and substratewere allowed to equilibrate for five minutes in a water bath shaker at30° C. Each reaction was initiated by injection of H₂ O₂ (20 μl of 1.0MH₂ O₂ in water) to a concentration of about 100 mM H₂ O₂. After anappropriate period (2 minutes) a portion of the reaction mixture wasremoved, the reaction terminated, and the sample prepared for analysis.

The hydroxylase remained active and catalyzed oxidation for multipleturnovers until the reactions were terminated. For example, 17 turnoversoccurred in 30 minutes when propene was used as a substrate at 10 mM H₂O₂. The hydroxylase was found to retain about 70% of its originalactivity upon completion of the run.

Samples were analyzed for products as follows: The benzene, substitutedbenzenes and pyridine samples were analyzed by High Pressure LiquidChromatography (HPLC). The reaction in each sample was quenched byaddition of 2 volumes of ethanol or 3% trichloroacetic acid which causedprecipitation of the hydroxylase. The samples were centrifuged for twominutes (10,800×g) and the precipitated hydroxylase removed. A portionof each supernatant was injected into a Beckman HPLC equipped with areverse-phase column (C₁₈ matrix) and UV detection capability.

All other samples were analyzed by gas chromatography. Except for themethane sample, all analyses were performed as follows: The reaction ineach sample was quenched by addition of an equal volume chloroform(CHCl₃) followed by vortexing for a brief period. The sample wascentrifuged for two minutes (10,800×g) to separate the CHCl₃ layer fromthe sample solution. A portion of the CHCl₃ layer was injected into aHewlett Packard 5890A gas chromatograph (100% methylsilicone columnmatrix) for analysis of the products.

For the methane sample, the reaction was quenched by addition of 0.1Macid to a pH of 2.9. The resulting aqueous phase was separated from thehydroxylase using a YM30 Centricon ultrafiltration unit. The filtratewas analyzed for methanol by gas chromatography.

Products resulting from the various substrates were identified bycomparison with standard compounds. Product distributions are presentedin Table VI.

                                      TABLE I                                     __________________________________________________________________________    SUMMARY OF THE PURIFICATION OF METHANE MONOOXYGENASE                          FROM METHYLOSINUS TRICHOSPORIUM OB3b.sup.a                                                      Total                                                                             Total                                                                              Specific                                                        Volume                                                                             Protein                                                                           Activity                                                                           Activity                                                                            Yield                                                                             Fold                                     Step         ml   mg  mUnits.sup.b                                                                       mUnits/mg                                                                           %   Purification                             __________________________________________________________________________    Hydroxylase                                                                   Cell free extract                                                                          630  11150                                                                             2230000                                                                             200  100 1.0                                      DEAE-Sepharose CL-6B                                                                       177  1590                                                                              1427000                                                                             900  64  4.5                                      Sephacryl S-300                                                                             95  835 1420000                                                                            1700  63  8.5                                      Component B                                                                   Cell free extract                                                                          630  11150                                                                             2230000                                                                             200  100 1.0                                      DEAE Sepharose CL-6B                                                                       169  590 1410000                                                                            2400  63  12.0                                     Sephadex G-50                                                                              118  153 2563000                                                                            16700 115 83.5.sup.c                               DEAE Sepharose CL-6B                                                                        62  110 1237000                                                                            11200 55  56.0                                     Reductase                                                                     Cell free extract                                                                          630  11150                                                                              835000                                                                             75   100 1.0                                      DEAE Sepharose CL-6B                                                                       160  136  708000                                                                            5200  85  69.3                                     DEAE Sepharose CL-6B                                                                        68   68  651000                                                                            9600  78  128.0                                    Ultrogel AcA-54                                                                             18   21  550000                                                                            26100 66  348.0                                    __________________________________________________________________________     .sup.a Results from separate purifications for each component were            normalized to the total protein in the cell free extract obtained from 20     g cell paste.                                                                 .sup.b A unit is defined as the production of 1 μmol of propene oxide      per minute.                                                                   .sup.c The further purification step is required to remove trace              cytochrome contaminants.                                                 

                  TABLE II                                                        ______________________________________                                        PHYSICAL PROPERTIES OF PURIFIED                                               METHANE MONOOXYGENASES                                                                Methylosinus                                                                           Methylococcus                                                                             Methylobacterium                                         trichosporium                                                                          capsulatus species                                                   OB3b.sup.a                                                                             (Bath).sup.b,c                                                                           CRL-26.sup.d,e                                    ______________________________________                                        Hydroxylase                                                                   s.sub.20,w                                                                              14.2       13.5       9.8                                           (sec × 10.sup.13)                                                       D.sub.20,w (cm.sup.2 /                                                                   4.3                                                                sec × 10.sup.7)                                                         Stokes radius                                                                           50.3       49.2                                                     (Å)                                                                       Subunit   (αβγ).sub.2                                                             (αβγ).sub.2                                                             (αβγ).sub.2                  structure                                                                     Subunit molec-                                                                          54.4,43.0,22.7                                                                           54,42,17   55,40,20                                      ular weights                                                                  (kDa)                                                                         Molecular weight (kDa)                                                        sedimentation                                                                           252                   225                                           velocity                                                                      gel filtration                                                                          245        210        220                                           estimated from                                                                          241        226        230                                           SDS-PAGE                                                                      Fe content                                                                               4.3        2.3       2.8                                           (mol/mol)                                                                     Specific activity                                                                       1700        72        208                                           % recovery                                                                               63         8          80                                           mg obtained/                                                                            835        156        800                                           200 g cel paste                                                               Component B                                                                   s.sub.20,w                                                                               1.6                                                                (sec × 10.sup.13)                                                       D.sub.20,w (cm.sub.2 /                                                                  10.9                                                                sec × 10.sup.7)                                                         Stokes radius                                                                           19.6                                                                (Å)                                                                       Molecular weight (kDa)                                                        sedimentation                                                                           15.1                                                                velocity                                                                      gel filtration                                                                          15- 31      17                                                      SDS-PAGE  15.8       15.7                                                     Metal content                                                                           none       none                                                     Specific activity                                                                       11200      7300                                                     pI         4.3        4                                                       % recovery                                                                               55         36                                                      mg obtained/                                                                            110         20                                                      200 g cell paste                                                              Reductase                                                                     s.sub.20,w                                                                               3.2                  2.1                                           (sec × 10.sup.13)                                                       D.sub.20,w (cm.sub.2 /                                                                   8.2                                                                sec × 10.sup.7)                                                         Stokes radius                                                                           26.2                                                                (Å)                                                                       Molecular weight (kDA)                                                        sedimentation                                                                           38.4                   38                                           velocity                                                                      gel filtration                                                                          38.3       44.6        40                                           SDS-PAGE  39.7        39         40                                           FAD        1          1          1                                            (mol/mol)                                                                     Fe content                                                                               2          2          2                                            (mol/mol)                                                                     Inorganic S                                                                              2          2          2                                            content                                                                       (mol/mol)                                                                     Specific activity                                                                       26100      6000       6200                                          % recovery                                                                               66         35         48                                           mg obtained/                                                                             21        248         18                                           200 g cell paste                                                              ______________________________________                                         .sup.a Typical cell yield 18-25 g cell paste per liter of culture media       .sup.b Typical cell yield estimated to be 20 g cell paste per liter of        culture media                                                                 .sup.c See Woodland et al (J. Biol. Chem. 259, 53-59, 1984), Green et al.     (J. Biol. Chem. 260, 15795-15801, 1985) and Colby et al. (Biochem. J. 177     903-908, (1979) for methods used to determine physical properties             .sup.d Typical cell yield 2-3 g cell paste per liter of culture media         .sup.e See Patel et al. (J. Bact. 169, 2313-2317, 1987) and Patel (Arch.      Biochem. Biophys. 252, 229-236, 1987) for methods used to determine           physical properties                                                      

                  TABLE III                                                       ______________________________________                                        IRON CONTENT OF THE METHANE                                                   MONOOXYGENASE HYDROXYLASE                                                           Specific Total   Mossbauer                                                                             EPR      EPR                                   Prep- Activity Iron.sup.a                                                                            [oxo-   mixed valent                                                                           g = 15                                ara-  nmol/    mol/    center]/                                                                              signal.sup.c                                                                           signal.sup.e                          tion  min/mg   mol     [protein].sup.b                                                                       spins/mol                                                                              mm                                    ______________________________________                                        1      70      1.9     0.91    0.66     13.5                                  2      70      2.1     --       0.07*   13.2                                  3      300     2.3     0.82    0.45     19.3                                  4      525     7.6     0.80    0.33     --                                    5      900     4.3     1.56    --       --                                    6     1000     4.8     --      .sup. 1.30.sup.e                                                                       22.6                                  7     1500     5.5     --      0.15     27.3                                  8     1700     4.3     1.79    0.52     33.1                                  ______________________________________                                         .sup.a Measure colorimetrically by complexation with                          2,4,6tripyridyl-s-triazine                                                    .sup.b Estimated from the intensity of the diamagentic material present i     the oxidized hydroxylase prepared from M. trichosporium OB3b cells grown      on media enriched with .sup.57 Fe (2.3 mg/liter) as observed by Mossbauer     spectroscopy, unreported samples were .sup.56 Fe hydroxylase preparations     .sup.c Measured by double integration of the mixed valent signal produced     by chemical reduction with sodium dithionite, except for preparations         marked "*," which were produced by NADH reduction in the presence of          catalytic amounts of component B and reductase                                .sup.d Estimated by measurement of the peak to trough displacement of the     g = 15 signal observed upon complete reduction                                .sup.e From five samples, the range of values was 0.6 to 1.3 spins per mo     with an average value of 0.70; for NADH reduction, 0.13 spin per mol was      observed                                                                 

                  TABLE IV(A)                                                     ______________________________________                                        SINGLE TURNOVER REACTIONS CATALYZED BY                                        THE HYDROXYLASE COMPONENT OF                                                  METHANE MONOOXYGENASE                                                                     Hydroxylation                                                                 of Propene Hydroxylation                                                      nmol           of Propane                                                     propene                                                                              %       nmol      %                                                    oxide  yield.sup.2                                                                           1-propanol                                                                              yield.sup.2                              ______________________________________                                        Reduced hydroxylase.sup.1                                                                    80.2     40.1    20.3    10.1                                  plus 200 nmol  83.8     41.9     9.0     4.5                                  component B                                                                   plus 50 nmol reductase                                                                       93.0     46.5   NA*     --                                     Mixed valent    6.9      6.9     2.9     2.9                                  hydroxylase                                                                   plus 200 nmol   7.4      7.4     3.6     3.6                                  component B                                                                   Redox mediators alone                                                                       0        0       0       0                                      Heat precipitated                                                                           0        0       0       0                                      hydroxylase                                                                   50 nmol reductase                                                                           0        0       NA      --                                     200 nmol component B                                                                        0        0       0       0                                      Hydroxylase, 500 μmol                                                                    0        0       0       0                                      NADH                                                                          ______________________________________                                         Single turnover experiments were performed as described in the                "Experimental Procedures."-                                                   *NA--not attempted                                                            .sup.1 100 nmol hydroxylase with specific activity of 1000 mUnits/mg.         .sup.2 Yield is calculated per binuclear iron center. The high activity       hydroxylase used here was determined to have two centers (four irons per      mol) (see Table III).                                                    

                  TABLE IV(B)                                                     ______________________________________                                        REACTIONS CATALYZED BY THE HYDROXYLASE                                        COMPONENT OF METHANE MONOOXYGENASE                                                       Hydroxylation                                                                             Hydroxylation                                                     of Methane  of Benzene                                                        nmol   %        nmol     %                                                    methanol                                                                             yield.sup.2                                                                            phenol   yield.sup.2                               ______________________________________                                        Reduced hydroxylase.sup.1                                                     340 nmol     61       18                                                      400 nmol                       14     3.5                                     ______________________________________                                         All experiments were performed as described in the "Experimental              Procedures."-                                                                 *NA--not attempted                                                            .sup.1 Specific activity 155 mUnits/mg                                        .sup.2 Yield is calculated per binuclear iron center. The low activity        hydroxylase used here was determined to have one center (two irons per        mol) (see Table III).                                                    

                  TABLE V                                                         ______________________________________                                        MULTIPLE TURNOVER REACTIONS CATALYZED                                         BY THE HYDROXYLASE COMPONENT OF                                               METHANE MONOOXYGENASE                                                         Specific Activity                                                                              Hydroxylation of Propene                                     Cycle  mUnits/mg     nmol propene oxide                                                                          % yield.sup.1                              ______________________________________                                        --     151           --            --                                         1      152           116           34                                         2      162           126           71                                         7      ND*           533           157                                        ______________________________________                                         Multiple turnover experiments were performed as in the "Experimental          Procedures."-                                                                 .sup.1 Yield is calculated per binuclear iron center. The low activity        hydroxylase used here was determined to have about one center (two irons      per mol) (see Table III).                                                     *ND--Not determined                                                      

                  TABLE VI                                                        ______________________________________                                        PRODUCT YIELD FROM H.sub.2 O.sub.2                                            COUPLED METHANE MONOOXYGENASE                                                 CATALYZED REACTIONS                                                                                      Relative Distribution                              Substrate   Products       (Percent)                                          ______________________________________                                        Methane     Methanol       100                                                Ethane      Ethanol        100                                                Propane     1-propanol     15                                                             2-propanol     85                                                 Propene     Propylene Oxide                                                                              100                                                Butane      1-butanol      64                                                             2-butanol      36                                                 Isobutane   2-methyl-1-propanol                                                                          61                                                             2-methyl-2-propanol                                                                          39                                                 Isopentane  2-methyl-1-butanol                                                                           25                                                             3-methyl-1-butanol                                                                           11                                                             3-methyl-2-butanol                                                                           24                                                             2-methyl-2-butanol                                                                           41                                                 1,1-dimethyl-                                                                             Methylcyclopropyl-                                                                           86                                                 cyclopropane                                                                              methanol                                                                      1-methylcyclobutanol                                                                         14                                                             3-methyl-3-buten-1-01                                                                        none detected                                      Cyclohexane Cyclohexanol   100                                                Benzene     Phenol         100                                                Nitrobenzene                                                                              p-Nitrophenol  42                                                             m-Nitrophenol  53                                                             o-Nitrophenol   5                                                 Pyridine    Pyridine-N-oxide                                                                             100                                                ______________________________________                                         Hydroxylation experiments were performed as described in "Experimental        Procedures"-                                                             

That which is claimed is:
 1. A method for oxidizing hydrocarbons in theabsence of reductase and B components of a soluble methane monooxygenasecomprising contacting purified hydroxylase from a soluble methanemonooxygenase with said hydrocarbons and hydrogen peroxide until atleast a portion of the corresponding oxidized product is produced in anisolable amount.
 2. The method for oxidizing hydrocarbons of claim 1wherein said hydroxylase contains at least about 3.5 mols of iron permol of protein.
 3. The method for oxidizing hydrocarbons of claim 2wherein said hydroxylase is obtained from the bacterium Methylosinustrichosporium OB3b.
 4. The method for oxidizing hydrocarbons of claim 1wherein said hydroxylase contains at least about 4.0 mols of iron permol of protein.
 5. The method for oxidizing hydrocarbons of claim 1wherein said hydroxylase has a final specific activity of at least about800 nmol/min/mg or greater.
 6. The method for oxidizing hydrocarbons ofclaim 5 wherein said hydroxylase is obtained from the bacteriumMethylosinus trichosporium OB3b.
 7. The method for oxidizinghydrocarbons of claim 1 wherein said hydroxylase has a final specificactivity of at least about 1000 nmol/min/mg or greater.
 8. A method foroxidizing hydrocarbons in the absence of reductase and B components of asoluble methane monooxygenase comprising contacting said hydrocarbonswith hydrogen peroxide and a catalyst consisting essentially of purifiedhydroxylase from a soluble methane monooxygenase until at least aportion of the corresponding oxidized product is produced in an isolableamount.